CLIC4和14-3-3蛋白在饥饿诱导神经胶质瘤自噬中的作用机制
摘要
恶性胶质瘤在成年人的脑部肿瘤中最为常见,发病率超过50%以上。虽然应用多种常规治疗手段,例如手术,射线治疗,化学治疗等,但是初诊之后患者的平均寿命一般都低于一年。尽管caspase介导的凋亡是抑制肿瘤生长的最有效的细胞死亡程序,但胶质瘤却能够抵御常规的促凋亡性的癌症治疗策略。因此,有效的治疗恶性胶质瘤很可能会涉及到诱导非凋亡性的细胞死亡,例如自噬性细胞死亡或者是有丝分裂突变性的细胞死亡,在最近的研究中被广泛认为是新兴的可选择的细胞死亡通路。
自噬,一种调节细胞内容物降解和再循环的进程,也能够参与器官的再生以及饥饿下的生物能学调控。在自噬情况下,部分的胞浆以及细胞器会被引入一个双层膜结构的称作自噬空泡或者是自噬体的结构中。这些自噬体能够与溶酶体融合形成单层膜结构的自噬溶酶体并降解其内容物。目前的研究中,有两种可以作为自噬信号激活参照的蛋白,其一是Beclin 1,一种对于自噬十分重要的种系保守蛋白,是一种单倍不足(haplo insufficient)的肿瘤抑制因子,在class III PI3K途径的自噬激活时Beclin 1信号水平上调。通过杂合子干扰Beclin 1自噬基因能引起肿瘤发生[44]。LC3蛋白是另一种自噬标志物,胞浆中的LC3-I与磷脂酰乙醇胺共价结合形成LC3-II的形式,并参与自噬体膜的形成[51]。尽管自噬能够在饥饿情况下回收大分子以及清除受损的线粒体及其他细胞器,为细胞提供能量以及蛋白质,可以被看作是一种抗凋亡的自我保护机制。但是,自噬也可以引起生长阻滞,细胞数下降,以及一种与过量的自噬空泡形成有关的非凋亡性的细胞死亡。因此,高水平的自噬也可以看做是一种细胞死亡功能。有关自噬的分子机制以及自噬在细胞死亡中的作用和意义仍有很多不明确的部分,还需要进一步的深入研究和探讨。
细胞内氯通道CLIC家族最具代表性的成员就是CLIC4,它对于p53和c-Myc调节的凋亡是十分重要的,其启动子是这些转录因子的下游靶点。胞浆内的CLIC4在代谢应激、生长阻滞、凋亡或者DNA损伤的时候,会在其自身的核定位序列,即NLS调节下转位入核。CLIC4是一种参与多种生理进程的重要因子,包括血管形成、成纤维细胞的转移分化、以及脂肪细胞分化等都与其有密切关系[121, 122]。主要定位于线粒体膜上的CLIC4,作为唯一的定位于线粒体上的CLIC家族成员,CLIC4蛋白对于维持线粒体功能和结构的稳态也具有重要的意义。然而,CLIC4蛋白的功能还不十分明确,关于它在细胞死亡中的作用的研究也只是刚刚起步,尤其是CLIC4蛋白在细胞程序性死亡的另一种形式——细胞自噬中的作用,还未见相关报道。由于线粒体被广泛认可为细胞凋亡和自噬的中心环节,因此,能够影响线粒体功能的CLIC4与细胞凋亡密切相关的同时,是否也与细胞自噬有关,就成为了一个值得深入探讨和研究的新课题。
14-3-3蛋白家族是一个完全保守的螺旋二聚化丝氨酸/苏氨酸结合蛋白家族,能够结合多种蛋白受体。14-3-3蛋白与多种细胞进程有关,例如有丝分裂、DNA损伤、细胞周期调控、凋亡等,在恶性肿瘤的形成过程中扮演了重要的角色[124]。最近的研究表明,14-3-3蛋白的Tau亚型能够调节自噬相关蛋白Beclin 1表达水平,对于自噬有着不可或缺的调节作用[145],但是14-3-3蛋白的其他亚型是否也与自噬进程有关的关系还不明确。
本研究通过饥饿诱导人神经胶质瘤U251细胞发生自噬,利用siRNA技术部分沉默细胞内氯通道蛋白4 (CLIC4),探讨CLIC4及其相关的14-3-3蛋白在细胞自噬中的作用机制,以及程序性细胞死亡的两种主要形式自噬及凋亡之间的关系。
方法:
(1)人源神经胶质瘤U251细胞培养。
(2)根据CLIC4的基因序列以及RNAi设计原理,构建pSilencerTM3.1-H1 neo CLIC4 siRNA质粒并测序。通过RT-PCR、Western Blot等技术分析CLIC4 mRNA及蛋白表达水平。
(3)通过MTT法检测细胞生存率。Western Blot技术分析转染CLIC4 siRNA不同时间点24h和48h,U251细胞凋亡相关蛋白Bax、Bcl-2、cleaved caspase-3和胞浆cyt c的表达。利用MDC、LC3、Hoechst荧光染色观察细胞自噬水平变化。
(4)通过MDC、LC3、Hoechst、AO染色,共聚焦显微镜下观察饥饿诱导U251细胞自噬变化。同时通过Western Blot技术检测饥饿条件下U251细胞LC3蛋白水平;细胞凋亡相关蛋白Bax、Bcl-2、cleaved caspase-3和胞浆cyt c的表达。通过流式细胞术PI/Annexin-V FITC染色法,检测饥饿条件下细胞凋亡率变化。
(5)稳定转染CLIC4 siRNA的U251细胞,饥饿8h后,通过共聚焦显微镜观察MDC、LC3、Hoechst、AO染色结果,检测细胞自噬水平;Western Blot技术检测自噬相关蛋白LC3表达水平。
(6)通过Western Blot方法检测抑制CLIC4表达对于饥饿条件下细胞凋亡相关蛋白Bax、Bcl-2、cleaved caspase-3和胞浆cyt c的表达。通过流式细胞术检测饥饿条件下,稳定转染CLIC4 siRNA的U251细胞凋亡率变化。
(7)通过Hoechst、14-3-3 epsilon、CLIC4染色于共聚焦显微镜下观察抑制CLIC4表达对于饥饿条件下,14-3-3 epsilon蛋白与CLIC4共定位的影响。通过Western Blot技术检测Beclin 1及14-3-3蛋白表达。免疫共沉淀技术检测14-3-3 epsilon蛋白与CLIC4蛋白的结合水平。
(8)通过罗丹明123(Rho123)染色及流式细胞术检测稳定转染CLIC4 siRNA细胞在饥饿条件下的线粒体膜电势变化。RT-PCR检测抑制CLIC4表达后线粒体动力学相关的融合基因Mfn1、Mfn2、Opa1和分裂基因Fis1、MTP18 mRNA的表达水平。
结果:
(1)通过酶切鉴定和测序证明pSilencerTM3.1-H1 neo CLIC4 siRNA的重组质粒构建成功。与空质粒对照组相比,CLIC4表达下降并具有显著统计学意义。
(2)与对照组相比,抑制CLIC4 siRNA对于U251细胞生存率无明显影响。同时,对于U251细胞凋亡及自噬均无显著影响。
(3)饥饿8h能够诱导诱导U251细胞发生自噬,LC3 II蛋白水平显著增加。细胞凋亡相关蛋白及细胞凋亡率表达无明显变化。表明饥饿并不能引起细胞凋亡过程的发生。
(4)与单纯饥饿组相比,稳定转染CLIC4 siRNA细胞组饥饿8h,LC3蛋白积聚明显增强,细胞自噬空泡积聚增加,LC3 II蛋白水平增加,表明抑制CLIC4表达促进了饥饿诱导的U251细胞自噬。饥饿条件下,抑制CLIC4表达能够在促进细胞自噬的同时启动饥饿条件下的U251细胞caspase途径相关的细胞凋亡过程。
(5)饥饿条件下,14-3-3 epsilon蛋白与CLIC4共定位显著增加,表明CLIC4对于自噬的调节作用很可能与14-3-3蛋白有关,而抑制CLIC4表达能够显著降低两者的共定位。抑制CLIC4表达能够引起自噬相关蛋白Beclin 1以及14-3-3蛋白表达增加。饥饿条件下,14-3-3 epsilon蛋白与CLIC4共沉淀增强,而抑制CLIC4表达能够降低两者结合水平,促进了14-3-3蛋白水平上调,引起Beclin 1蛋白表达增加,激活自噬。
(6)抑制CLIC4表达引起了饥饿条件下的线粒体膜电势下降。与饥饿组相比,抑制CLIC4表达能够上调线粒体融合基因和分裂基因表达水平,引起线粒体动力学紊乱,导致线粒体过度损伤,这很可能是其促进细胞自噬并引发凋亡的另一原因。
结论:
本研究利用RNA干扰技术部分沉默主要定位于线粒体的细胞内氯通道蛋白4(CLIC4),通过饥饿诱导人源神经胶质瘤U251细胞发生自噬,探讨U251细胞自噬和凋亡的关系以及CLIC4和14-3-3蛋白在此过程中的作用。实验发现单纯的抑制CLIC4表达对于U251细胞自噬和凋亡均无显著影响。而饥饿条件下,U251细胞发生自噬的同时并没有明显的细胞凋亡过程。利用RNAi技术抑制CLIC4表达,促进了饥饿条件下的U251细胞自噬,同时,也能够启动线粒体相关的caspase途径的细胞凋亡。而动力学相关的线粒体损伤以及14-3-3蛋白与CLIC4相互作用的减弱,引起14-3-3蛋白表达上调,Beclin 1信号通路进一步的激活,很可能是引发这一系列反应的主要原因。
本研究首次探讨了饥饿诱导的自噬中主要定位于线粒体的细胞内氯通道蛋白4(CLIC4)以及14-3-3蛋白在这一过程中的作用。进一步探讨了胶质瘤细胞中自噬的作用机制以及自噬与凋亡这两种程序性细胞死亡的主要形式之间的关系,再次印证了线粒体是联系细胞自噬和凋亡的关键所在。深入研究自噬与凋亡的关系,将细胞自噬相关的治疗方法引入治疗策略之中,很可能会成为肿瘤治疗的一条新的途径。
Malignant Gliomas account for over 50% of all brain tumors and are the most common primary brain tumors in adults. Despite the use of conventional treatments, including surgery, irradiation therapy, and chemotherapy, the average life expectancy of glioma patients after the initial diagnosis is usually less than 1 year. Although caspase mediated apoptosis is the best-defined cell death program counteracting tumor growth, glioma cells are resistant to the conventional proapoptotic cancer therapeutics. Therefore, effective treatment of malignant gliomas may rely on the development of novel strategies for inducing nonapoptotic cell death, such as autophagic cell death or cell death through mitotic catastrophe, which has been recently described as a new alternative death pathway.
Autophagy, a regulated process of degradation and recycling of cellular constituents, also participates in organelle turnover and in the bioenergetic management of starvation. During autophagy, parts of the cytoplasm or entire organelles are sequestered into double-membraned vesicles called autophagic vacuoles or autophagosomes. These autophagosomes ultimately fuse with lysosomes to generate single-membraned autophago -lysosomes capable of degrading their contents. There are two main signal markers for the detection of autophagy, one is Beclin 1, a phylogenically conserved protein essential for autophagy, is a haplo insufficient tumor suppressor, activate and up-regulate in the autophagy through class III PI3K pathway. LC3 protein is another autophagy marker, cytosolic protein LC3-I covalently linked to phosphatidylethanolamine, then converts to LC3-II, associates with the formation of autophagosome. Although autophagy can serve as a protective mechanism against apoptosis and starvation by recycling macromolecules and removing damaged mitochondria and other organelles, it can also lead to growth arrest, reduction in cell number, and a nonapoptotic cell death associated with appearance of excessive autophagic vesicles. Therefore, high levels of autophagy can function as a cell death effector mechanism. Recently, induction of autophagy or autophagic cell death has been reported in several types of cancer cells in response to radiation or chemotherapy. But the molecular mechanisms of autophagy remain largely unknown, still need further investigations.
The best characterized of the CLIC family members is CLIC4, essential for p53 and c-Myc mediated apoptosis and its promoter is a direct downstream target of these transcription factors. Cytoplasmic CLIC4 translocates to the nucleus under conditions of metabolic stress, growth arrest, apoptosis and DNA damage, this is mediated by a functional nuclear localization signal (NLS). CLIC4 has emerged as a crucial player in many physiological processes, including tubular morphogenesis during angiogenesis, transdifferentiation of mammary fibroblasts to myofibroblasts, and adipocyte differentiation. CLIC4 locate on the mitochondrial membrane, as the only member which is locate on the mitochondria, CLIC4 is important for the maintaince of the mitochondria founction and structure, to affect the founction of CLIC4 by genetic of biological techniques, maybe affect the normal mitochondria founction, and cell differentiation, generation and cell death urther. Mitochondria is regarded as the key of cell apoptosis and autophagy, so whether the CLIC4 protein which is relate to the cell apoptosis, is also relate to the autophagy, become a new subject that is great worth to discuss and research.
14-3-3 proteins are a family of about 30kDa dimeric well conserved a-helical phosphoserine/threonine binding proteins. They are able to bind multiple protein ligands. 14-3-3 protein is involved in many different cellular processes, including mitogenesis, DNA damage, cell cycle control, and apoptosis, plays a significant role in the formation of malignant tumours. Latest research indicated that 14-3-3 protein isoform Tau regulated the level of Beclin1 and required for autophagy, but whether the other forms of 14-3-3 family are involved in autophagy process still not clear.
In this study, we induced autophagy in human glioma U251 cell line by starvation, partially silenced chloride intracellular channel protein 4 (CLIC4) with small interfere RNA (siRNA), investigate the mechanism of CLIC4 and 14-3-3 protein in autophagy, and the relationship between autophagy and apotosis which are the two main forms of programmed cell death.
Methods:
(1) Human glioma U251 cell culture.
(2) Base on the gene sequence and the RNAi desine principles, pSilencerTM3.1-H1 neo CLIC4 siRNA plasmid was constructed and sequencing. The level of CLIC4 mRNA and protein expression was analyzed by RT-PCR and Western Blot.
(3) To detect cell vitality, MTT method was applied. Fluorescence staining of MDC、LC3、 Hoechst to determine the change of cell autophagy. The levels of Bax、Bcl-2、cleaved caspase-3 and cytosolic cyt c protein were detected by Western blot, investigate the effect of the inhibition of CLIC4 on cell apoptosis.
(4) Fluorescence staining of MDC、LC3、Hoechst and AO to determine the autophagy under starvation. The levels of apoptotic protein Bax、Bcl-2、cleaved caspase-3 and cytosolic cyt c were detected by Western blot. The cell apoptosis rate was detected with PI/Annexin-V FITC staining by flow cytometry.
(5) To investigate the effect of the inhibition of CLIC4 on the autophagy induced by starvation, fluorescence staining of MDC、LC3、Hoechst and AO was applied for the autophagy detection, and Western Blot analysis to determine the LC3 protein level.
(6) To investigate the effect of the inhibition of CLIC4 on the apoptosis induced by starvation. The levels of apoptotic protein Bax、Bcl-2、cleaved caspase-3 and cytosolic cyt c were detected by Western blot. The cell apoptosis rate was detected by flow cytometry.
(7) To investigate the effect of the inhibition of CLIC4 on the co-location of 14-3-3 epsilon protein and CLIC4, fluorescence staining of Hoechst、14-3-3 epsilon、CLIC4 was used and Western Blot analysis to determine the expression level of Beclin 1 and 14-3-3 epsilon protein. Immunoprecipitation was applied to detect the binding of 14-3-3 protein and CLIC4 protein.
(8) Mitochondrial membrane potential was measured with Rhodamine123 (Rho123) staining and Flow cytometry analysis. The effect of CLIC4 siRNA on the mitochondrial fusion genes Mfn1, Mfn2, Opa1 and fission genes Fis1, MTP18 were determined by RT-PCR.
Results:
(1) We constructed recombinant plasmid of pSilencerTM3.1-H1 neo CLIC4 siRNA successfully, confirmed by restrictive enzyme digestion and DNA sequencing. The level of CLIC4 mRNA and protern in U251 cells decreased significantly.
(2) Compared with the control group, MTT results indicated that there is no obvious effect of cell vitality with CLIC4 siRNA transfection. The inhibition of CLIC4 has no significant effect on cell apoptosis and autophagy in U251 cells.
(3) Compared with control group, the expression of autophagy-associated protein LC3 II was up-regulated significantly. No change of apoptosis related protein expression. Starvation of U251 cells could not lead to cell apoptosis.
(4) The inhibition of CLIC4 enhanced cell autophagy, the accumulation of LC3 and cell autophagic vacuoles increased, LC3 II protein expression increased in U251 cells induced by starvation after 8 hours, compared with starvation group. Inhibition of CLIC4 triggered apoptosis in U251 cells under starvation.
(5) The results of 14-3-3 epsilon and CLIC4 fluo-staining indicated that the co-location of 14-3-3 epsilon protein and ClIC4 increased significantly under starvation, showed that the regulation of CLIC4 on autophagy is relate to the 14-3-3 epsilon protein probably, and the inhibition of CLIC4 down-regulated the co-location of them. The results of Western Blot indicated that inhibition of CLIC4 caused the increasing of Beclin 1 and 14-3-3 protein expression. The co-precipitation of 14-3-3 epsilon protein and CLIC4 enhanced under starvation was decreased by inhibition of CLIC4. In a word, the inhibition of the interaction between 14-3-3 epsilon protein and CLIC4 by siRNA, and the following over activation of Beclin 1 signal pathway, maybe the reason of the inhibition of CLIC4 aggravated the autophagy under starvation.
(6) The inhibition of CLIC4 decreased mitochondrial membrane potential, resulted mitochondrial injury and up-regulated mitochondrial fusion and fission genes,lead to mitochondria kinesics disorder, maybe the another reason of autophagy and apoptosis exacerbation.
Conclusions:
This research partially silenced mitochondria localized chloride intracellular channel protein 4 (CLIC4) with small interfere RNA (siRNA), induced human glioma U251 cell autophagy by starvation, to investigate the relation of autophagy and apoptosis, and the machenism of CLIC4 and 14-3-3 protein in this process. The results showed that inhibition of CLIC4 has no effect on cell autophagy and apoptosis. Under starvation, autophagy was detected in U251 cells but no apoptosis. Inhibition of CLIC4 enhanced the autophagy under starvation, on the same time, triggered caspase-dependent mitochondrial apoptosis. The mitochondrial dynamic related injury and decrease of the interaction between 14-3-3 protein and CLIC4, over activated Beclin 1 signaling pathway, maybe the reasons of these chain reactions.
This study investigated the effect of choloride intracellular channel protein 4(CLIC4) which is located on mitochondria mainly and 14-3-3 protein in the autophagy induced by starvation for the first time. Further investigated the mechanism of cell autophagy in glioma cells, and the relationship between autophagy and apotosis which are the two main forms of programmed cell death, indicated that mitochondria is the key to connect autophagy and apoptosis. The further research on the relation of autophagy and apoptosis, introduce autophagy related methods into the therapic strategy, maybe the new route of cancer therapy.
自噬,一种调节细胞内容物降解和再循环的进程,也能够参与器官的再生以及饥饿下的生物能学调控。在自噬情况下,部分的胞浆以及细胞器会被引入一个双层膜结构的称作自噬空泡或者是自噬体的结构中。这些自噬体能够与溶酶体融合形成单层膜结构的自噬溶酶体并降解其内容物。目前的研究中,有两种可以作为自噬信号激活参照的蛋白,其一是Beclin 1,一种对于自噬十分重要的种系保守蛋白,是一种单倍不足(haplo insufficient)的肿瘤抑制因子,在class III PI3K途径的自噬激活时Beclin 1信号水平上调。通过杂合子干扰Beclin 1自噬基因能引起肿瘤发生[44]。LC3蛋白是另一种自噬标志物,胞浆中的LC3-I与磷脂酰乙醇胺共价结合形成LC3-II的形式,并参与自噬体膜的形成[51]。尽管自噬能够在饥饿情况下回收大分子以及清除受损的线粒体及其他细胞器,为细胞提供能量以及蛋白质,可以被看作是一种抗凋亡的自我保护机制。但是,自噬也可以引起生长阻滞,细胞数下降,以及一种与过量的自噬空泡形成有关的非凋亡性的细胞死亡。因此,高水平的自噬也可以看做是一种细胞死亡功能。有关自噬的分子机制以及自噬在细胞死亡中的作用和意义仍有很多不明确的部分,还需要进一步的深入研究和探讨。
细胞内氯通道CLIC家族最具代表性的成员就是CLIC4,它对于p53和c-Myc调节的凋亡是十分重要的,其启动子是这些转录因子的下游靶点。胞浆内的CLIC4在代谢应激、生长阻滞、凋亡或者DNA损伤的时候,会在其自身的核定位序列,即NLS调节下转位入核。CLIC4是一种参与多种生理进程的重要因子,包括血管形成、成纤维细胞的转移分化、以及脂肪细胞分化等都与其有密切关系[121, 122]。主要定位于线粒体膜上的CLIC4,作为唯一的定位于线粒体上的CLIC家族成员,CLIC4蛋白对于维持线粒体功能和结构的稳态也具有重要的意义。然而,CLIC4蛋白的功能还不十分明确,关于它在细胞死亡中的作用的研究也只是刚刚起步,尤其是CLIC4蛋白在细胞程序性死亡的另一种形式——细胞自噬中的作用,还未见相关报道。由于线粒体被广泛认可为细胞凋亡和自噬的中心环节,因此,能够影响线粒体功能的CLIC4与细胞凋亡密切相关的同时,是否也与细胞自噬有关,就成为了一个值得深入探讨和研究的新课题。
14-3-3蛋白家族是一个完全保守的螺旋二聚化丝氨酸/苏氨酸结合蛋白家族,能够结合多种蛋白受体。14-3-3蛋白与多种细胞进程有关,例如有丝分裂、DNA损伤、细胞周期调控、凋亡等,在恶性肿瘤的形成过程中扮演了重要的角色[124]。最近的研究表明,14-3-3蛋白的Tau亚型能够调节自噬相关蛋白Beclin 1表达水平,对于自噬有着不可或缺的调节作用[145],但是14-3-3蛋白的其他亚型是否也与自噬进程有关的关系还不明确。
本研究通过饥饿诱导人神经胶质瘤U251细胞发生自噬,利用siRNA技术部分沉默细胞内氯通道蛋白4 (CLIC4),探讨CLIC4及其相关的14-3-3蛋白在细胞自噬中的作用机制,以及程序性细胞死亡的两种主要形式自噬及凋亡之间的关系。
方法:
(1)人源神经胶质瘤U251细胞培养。
(2)根据CLIC4的基因序列以及RNAi设计原理,构建pSilencerTM3.1-H1 neo CLIC4 siRNA质粒并测序。通过RT-PCR、Western Blot等技术分析CLIC4 mRNA及蛋白表达水平。
(3)通过MTT法检测细胞生存率。Western Blot技术分析转染CLIC4 siRNA不同时间点24h和48h,U251细胞凋亡相关蛋白Bax、Bcl-2、cleaved caspase-3和胞浆cyt c的表达。利用MDC、LC3、Hoechst荧光染色观察细胞自噬水平变化。
(4)通过MDC、LC3、Hoechst、AO染色,共聚焦显微镜下观察饥饿诱导U251细胞自噬变化。同时通过Western Blot技术检测饥饿条件下U251细胞LC3蛋白水平;细胞凋亡相关蛋白Bax、Bcl-2、cleaved caspase-3和胞浆cyt c的表达。通过流式细胞术PI/Annexin-V FITC染色法,检测饥饿条件下细胞凋亡率变化。
(5)稳定转染CLIC4 siRNA的U251细胞,饥饿8h后,通过共聚焦显微镜观察MDC、LC3、Hoechst、AO染色结果,检测细胞自噬水平;Western Blot技术检测自噬相关蛋白LC3表达水平。
(6)通过Western Blot方法检测抑制CLIC4表达对于饥饿条件下细胞凋亡相关蛋白Bax、Bcl-2、cleaved caspase-3和胞浆cyt c的表达。通过流式细胞术检测饥饿条件下,稳定转染CLIC4 siRNA的U251细胞凋亡率变化。
(7)通过Hoechst、14-3-3 epsilon、CLIC4染色于共聚焦显微镜下观察抑制CLIC4表达对于饥饿条件下,14-3-3 epsilon蛋白与CLIC4共定位的影响。通过Western Blot技术检测Beclin 1及14-3-3蛋白表达。免疫共沉淀技术检测14-3-3 epsilon蛋白与CLIC4蛋白的结合水平。
(8)通过罗丹明123(Rho123)染色及流式细胞术检测稳定转染CLIC4 siRNA细胞在饥饿条件下的线粒体膜电势变化。RT-PCR检测抑制CLIC4表达后线粒体动力学相关的融合基因Mfn1、Mfn2、Opa1和分裂基因Fis1、MTP18 mRNA的表达水平。
结果:
(1)通过酶切鉴定和测序证明pSilencerTM3.1-H1 neo CLIC4 siRNA的重组质粒构建成功。与空质粒对照组相比,CLIC4表达下降并具有显著统计学意义。
(2)与对照组相比,抑制CLIC4 siRNA对于U251细胞生存率无明显影响。同时,对于U251细胞凋亡及自噬均无显著影响。
(3)饥饿8h能够诱导诱导U251细胞发生自噬,LC3 II蛋白水平显著增加。细胞凋亡相关蛋白及细胞凋亡率表达无明显变化。表明饥饿并不能引起细胞凋亡过程的发生。
(4)与单纯饥饿组相比,稳定转染CLIC4 siRNA细胞组饥饿8h,LC3蛋白积聚明显增强,细胞自噬空泡积聚增加,LC3 II蛋白水平增加,表明抑制CLIC4表达促进了饥饿诱导的U251细胞自噬。饥饿条件下,抑制CLIC4表达能够在促进细胞自噬的同时启动饥饿条件下的U251细胞caspase途径相关的细胞凋亡过程。
(5)饥饿条件下,14-3-3 epsilon蛋白与CLIC4共定位显著增加,表明CLIC4对于自噬的调节作用很可能与14-3-3蛋白有关,而抑制CLIC4表达能够显著降低两者的共定位。抑制CLIC4表达能够引起自噬相关蛋白Beclin 1以及14-3-3蛋白表达增加。饥饿条件下,14-3-3 epsilon蛋白与CLIC4共沉淀增强,而抑制CLIC4表达能够降低两者结合水平,促进了14-3-3蛋白水平上调,引起Beclin 1蛋白表达增加,激活自噬。
(6)抑制CLIC4表达引起了饥饿条件下的线粒体膜电势下降。与饥饿组相比,抑制CLIC4表达能够上调线粒体融合基因和分裂基因表达水平,引起线粒体动力学紊乱,导致线粒体过度损伤,这很可能是其促进细胞自噬并引发凋亡的另一原因。
结论:
本研究利用RNA干扰技术部分沉默主要定位于线粒体的细胞内氯通道蛋白4(CLIC4),通过饥饿诱导人源神经胶质瘤U251细胞发生自噬,探讨U251细胞自噬和凋亡的关系以及CLIC4和14-3-3蛋白在此过程中的作用。实验发现单纯的抑制CLIC4表达对于U251细胞自噬和凋亡均无显著影响。而饥饿条件下,U251细胞发生自噬的同时并没有明显的细胞凋亡过程。利用RNAi技术抑制CLIC4表达,促进了饥饿条件下的U251细胞自噬,同时,也能够启动线粒体相关的caspase途径的细胞凋亡。而动力学相关的线粒体损伤以及14-3-3蛋白与CLIC4相互作用的减弱,引起14-3-3蛋白表达上调,Beclin 1信号通路进一步的激活,很可能是引发这一系列反应的主要原因。
本研究首次探讨了饥饿诱导的自噬中主要定位于线粒体的细胞内氯通道蛋白4(CLIC4)以及14-3-3蛋白在这一过程中的作用。进一步探讨了胶质瘤细胞中自噬的作用机制以及自噬与凋亡这两种程序性细胞死亡的主要形式之间的关系,再次印证了线粒体是联系细胞自噬和凋亡的关键所在。深入研究自噬与凋亡的关系,将细胞自噬相关的治疗方法引入治疗策略之中,很可能会成为肿瘤治疗的一条新的途径。
Malignant Gliomas account for over 50% of all brain tumors and are the most common primary brain tumors in adults. Despite the use of conventional treatments, including surgery, irradiation therapy, and chemotherapy, the average life expectancy of glioma patients after the initial diagnosis is usually less than 1 year. Although caspase mediated apoptosis is the best-defined cell death program counteracting tumor growth, glioma cells are resistant to the conventional proapoptotic cancer therapeutics. Therefore, effective treatment of malignant gliomas may rely on the development of novel strategies for inducing nonapoptotic cell death, such as autophagic cell death or cell death through mitotic catastrophe, which has been recently described as a new alternative death pathway.
Autophagy, a regulated process of degradation and recycling of cellular constituents, also participates in organelle turnover and in the bioenergetic management of starvation. During autophagy, parts of the cytoplasm or entire organelles are sequestered into double-membraned vesicles called autophagic vacuoles or autophagosomes. These autophagosomes ultimately fuse with lysosomes to generate single-membraned autophago -lysosomes capable of degrading their contents. There are two main signal markers for the detection of autophagy, one is Beclin 1, a phylogenically conserved protein essential for autophagy, is a haplo insufficient tumor suppressor, activate and up-regulate in the autophagy through class III PI3K pathway. LC3 protein is another autophagy marker, cytosolic protein LC3-I covalently linked to phosphatidylethanolamine, then converts to LC3-II, associates with the formation of autophagosome. Although autophagy can serve as a protective mechanism against apoptosis and starvation by recycling macromolecules and removing damaged mitochondria and other organelles, it can also lead to growth arrest, reduction in cell number, and a nonapoptotic cell death associated with appearance of excessive autophagic vesicles. Therefore, high levels of autophagy can function as a cell death effector mechanism. Recently, induction of autophagy or autophagic cell death has been reported in several types of cancer cells in response to radiation or chemotherapy. But the molecular mechanisms of autophagy remain largely unknown, still need further investigations.
The best characterized of the CLIC family members is CLIC4, essential for p53 and c-Myc mediated apoptosis and its promoter is a direct downstream target of these transcription factors. Cytoplasmic CLIC4 translocates to the nucleus under conditions of metabolic stress, growth arrest, apoptosis and DNA damage, this is mediated by a functional nuclear localization signal (NLS). CLIC4 has emerged as a crucial player in many physiological processes, including tubular morphogenesis during angiogenesis, transdifferentiation of mammary fibroblasts to myofibroblasts, and adipocyte differentiation. CLIC4 locate on the mitochondrial membrane, as the only member which is locate on the mitochondria, CLIC4 is important for the maintaince of the mitochondria founction and structure, to affect the founction of CLIC4 by genetic of biological techniques, maybe affect the normal mitochondria founction, and cell differentiation, generation and cell death urther. Mitochondria is regarded as the key of cell apoptosis and autophagy, so whether the CLIC4 protein which is relate to the cell apoptosis, is also relate to the autophagy, become a new subject that is great worth to discuss and research.
14-3-3 proteins are a family of about 30kDa dimeric well conserved a-helical phosphoserine/threonine binding proteins. They are able to bind multiple protein ligands. 14-3-3 protein is involved in many different cellular processes, including mitogenesis, DNA damage, cell cycle control, and apoptosis, plays a significant role in the formation of malignant tumours. Latest research indicated that 14-3-3 protein isoform Tau regulated the level of Beclin1 and required for autophagy, but whether the other forms of 14-3-3 family are involved in autophagy process still not clear.
In this study, we induced autophagy in human glioma U251 cell line by starvation, partially silenced chloride intracellular channel protein 4 (CLIC4) with small interfere RNA (siRNA), investigate the mechanism of CLIC4 and 14-3-3 protein in autophagy, and the relationship between autophagy and apotosis which are the two main forms of programmed cell death.
Methods:
(1) Human glioma U251 cell culture.
(2) Base on the gene sequence and the RNAi desine principles, pSilencerTM3.1-H1 neo CLIC4 siRNA plasmid was constructed and sequencing. The level of CLIC4 mRNA and protein expression was analyzed by RT-PCR and Western Blot.
(3) To detect cell vitality, MTT method was applied. Fluorescence staining of MDC、LC3、 Hoechst to determine the change of cell autophagy. The levels of Bax、Bcl-2、cleaved caspase-3 and cytosolic cyt c protein were detected by Western blot, investigate the effect of the inhibition of CLIC4 on cell apoptosis.
(4) Fluorescence staining of MDC、LC3、Hoechst and AO to determine the autophagy under starvation. The levels of apoptotic protein Bax、Bcl-2、cleaved caspase-3 and cytosolic cyt c were detected by Western blot. The cell apoptosis rate was detected with PI/Annexin-V FITC staining by flow cytometry.
(5) To investigate the effect of the inhibition of CLIC4 on the autophagy induced by starvation, fluorescence staining of MDC、LC3、Hoechst and AO was applied for the autophagy detection, and Western Blot analysis to determine the LC3 protein level.
(6) To investigate the effect of the inhibition of CLIC4 on the apoptosis induced by starvation. The levels of apoptotic protein Bax、Bcl-2、cleaved caspase-3 and cytosolic cyt c were detected by Western blot. The cell apoptosis rate was detected by flow cytometry.
(7) To investigate the effect of the inhibition of CLIC4 on the co-location of 14-3-3 epsilon protein and CLIC4, fluorescence staining of Hoechst、14-3-3 epsilon、CLIC4 was used and Western Blot analysis to determine the expression level of Beclin 1 and 14-3-3 epsilon protein. Immunoprecipitation was applied to detect the binding of 14-3-3 protein and CLIC4 protein.
(8) Mitochondrial membrane potential was measured with Rhodamine123 (Rho123) staining and Flow cytometry analysis. The effect of CLIC4 siRNA on the mitochondrial fusion genes Mfn1, Mfn2, Opa1 and fission genes Fis1, MTP18 were determined by RT-PCR.
Results:
(1) We constructed recombinant plasmid of pSilencerTM3.1-H1 neo CLIC4 siRNA successfully, confirmed by restrictive enzyme digestion and DNA sequencing. The level of CLIC4 mRNA and protern in U251 cells decreased significantly.
(2) Compared with the control group, MTT results indicated that there is no obvious effect of cell vitality with CLIC4 siRNA transfection. The inhibition of CLIC4 has no significant effect on cell apoptosis and autophagy in U251 cells.
(3) Compared with control group, the expression of autophagy-associated protein LC3 II was up-regulated significantly. No change of apoptosis related protein expression. Starvation of U251 cells could not lead to cell apoptosis.
(4) The inhibition of CLIC4 enhanced cell autophagy, the accumulation of LC3 and cell autophagic vacuoles increased, LC3 II protein expression increased in U251 cells induced by starvation after 8 hours, compared with starvation group. Inhibition of CLIC4 triggered apoptosis in U251 cells under starvation.
(5) The results of 14-3-3 epsilon and CLIC4 fluo-staining indicated that the co-location of 14-3-3 epsilon protein and ClIC4 increased significantly under starvation, showed that the regulation of CLIC4 on autophagy is relate to the 14-3-3 epsilon protein probably, and the inhibition of CLIC4 down-regulated the co-location of them. The results of Western Blot indicated that inhibition of CLIC4 caused the increasing of Beclin 1 and 14-3-3 protein expression. The co-precipitation of 14-3-3 epsilon protein and CLIC4 enhanced under starvation was decreased by inhibition of CLIC4. In a word, the inhibition of the interaction between 14-3-3 epsilon protein and CLIC4 by siRNA, and the following over activation of Beclin 1 signal pathway, maybe the reason of the inhibition of CLIC4 aggravated the autophagy under starvation.
(6) The inhibition of CLIC4 decreased mitochondrial membrane potential, resulted mitochondrial injury and up-regulated mitochondrial fusion and fission genes,lead to mitochondria kinesics disorder, maybe the another reason of autophagy and apoptosis exacerbation.
Conclusions:
This research partially silenced mitochondria localized chloride intracellular channel protein 4 (CLIC4) with small interfere RNA (siRNA), induced human glioma U251 cell autophagy by starvation, to investigate the relation of autophagy and apoptosis, and the machenism of CLIC4 and 14-3-3 protein in this process. The results showed that inhibition of CLIC4 has no effect on cell autophagy and apoptosis. Under starvation, autophagy was detected in U251 cells but no apoptosis. Inhibition of CLIC4 enhanced the autophagy under starvation, on the same time, triggered caspase-dependent mitochondrial apoptosis. The mitochondrial dynamic related injury and decrease of the interaction between 14-3-3 protein and CLIC4, over activated Beclin 1 signaling pathway, maybe the reasons of these chain reactions.
This study investigated the effect of choloride intracellular channel protein 4(CLIC4) which is located on mitochondria mainly and 14-3-3 protein in the autophagy induced by starvation for the first time. Further investigated the mechanism of cell autophagy in glioma cells, and the relationship between autophagy and apotosis which are the two main forms of programmed cell death, indicated that mitochondria is the key to connect autophagy and apoptosis. The further research on the relation of autophagy and apoptosis, introduce autophagy related methods into the therapic strategy, maybe the new route of cancer therapy.
引文
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